Bait region cleavage and complex formation of human alpha M-2 with a Porphyromonas gingivalis W50 protease is not accompanied by enzyme inhibition

Three isoforms of extracellular Arg-specific proteases of P. gingivalis, W5 0, HRgpA, RgpA(cat) and mt-RgpA(cat), which are all products of the same ge ne, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to ex amine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (similar to 110 kDa) and RgpA(cat) (si milar to 55 kDa) with human alpha(2)M and their cytotoxicity toward culture d fibroblasts were investigated. Both enzymes formed complexes with alpha(2 )M as shown by gel filtration chromatography and both cleaved the 'bait' re gion at Arg(696)-Leu(697). However, whereas (alpha(2)M-RgpA(cat)) complex w as unable to hydrolyse large substrates such as hide powder azure, (alpha(2 )M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was abl e to bind to alpha(2)M saturated with trypsin and also to methylamine-treat ed alpha(2)M. This suggested that HRgpA is able to bind to both 'slow' and 'fast' forms of alpha(2)M and formation of (alpha(2)M:HRgpA) complex does n ot trap HRgpA and cause inhibition of activity toward hide powder azure. Ho wever, the (alpha(2)M-HRgpA) complex is not able to cleave other alpha(2)M molecules, which suggests that the active site of HRgpA in the complex is c onstrained probably due to steric reasons. The (alpha(2)M-HRgpA) complex wa s cytotoxic to 3T3 cells, causing them to round up and detach from the surf ace with a reduction in metabolic activity.